麦类作物原位杂交影响因素的研究

为了更好地利用原位杂交技术进行麦类作物外源染色体的检测和基因定位,对麦类作物原位杂交的影响因素进行了研究。（１）利用缺口转译法对克隆的ＤＮＡ 片段进行生物素标记,即节省时间,又可以获得较高的标记效率;对麦类作物的基因组ＤＮＡ,宜采用随机寡核苷酸引物标记法进行生物素标记,适当延长标记时间可以提高标记效率。（２）共变性法比较适宜麦类作物的原位杂交,分别变性法如掌握不当易使染色体产生膨胀现象,变性温度过高也会使黑麦（Ｓｅｃａｌｅ ｃｅｒｅａｌ）染色体的轮廓模糊不清。（３）应根据麦类作物亲本之间的亲缘关系决定封阻ＤＮＡ 的使用浓度,并利用生物素标记的簇毛麦（Ｈａｙｎａｌｄｉａ ｖｉｌｌｏｓａ）基因组ＤＮＡ 为探针,从普通小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）－簇毛麦双二倍体的染色体中识别了簇毛麦的染色体。（４）麦类作物的原位杂交受洗脱强度的影响很大,利用甲酰胺进行洗脱可以获得背景清晰的原位杂交带型。随着原位杂交技术分辨率的不断提高,该技术还将用于单拷贝基因的染色体定位     

原位杂交技术及其在果树研究中的应用

原位杂交技术是近年来快速发展起来的一门新技术,本文介绍了原位杂交技术的基本原理、方法及其发展前景,以及该技术与其它生物学技术相结合而形成的一些新技术。综述了这些技术在果树研究中的应用情况。     

原位杂交技术在植物遗传育种上的应用

本文在简要介绍原位杂交技术的基础之上 ,重点介绍了该技术在植物遗传育种领域 ,即在 (1 )异源染色质及染色体畸变检测 ;(2 )植物基因工程及基因表达研究 ;(3)构建植物基因物理图谱 ;(4)染色体RNA研究等方面的应用现状 ,并对原位杂交技术在提高检出率 ,与染色体显带技术结合 ,PCR 原位杂交等方面提了一些见解。     

Genetic Relationships Among Five Basic Genomes St,E,A,B and D in Triticeae Revealed by Genomic Southern and in situ Hybridization

The St and E are two important basic genomes in the perennial tribe Triticeae (Poaceae). They exist in many perennialspecies and are very closely related to the A, B and D genomes of bread wheat (Triticum aestivum L.). Genomic Southernhybridization and genomic in situ hybridization (GISH) were used to analyze the genomic relationships between the twogenomes (St and E) and the three basic genomes (A, B and D) of T. aestivum. The semi-quantitative analysis of the Southernhybridization suggested that both St and E genomes are most closely related to the D genome, then the A genome, andrelatively distant to the B genome. GISH analysis using St and E genomic DNA as probes further confirmed the conclusion.St and E are the two basic genomes of Thinopyrum ponticum (StStE~eE~bE~x) and Th. intermedium (StE~eE~b), two perennialspecies successfully used in wheat improvement. Therefore, this paper provides a possible answer as to why most of thespontaneous wheat-Thinopyrum translocations and substitutions usually happen in the D genome, some in the A genomeand rarely in the B genome. This would develop further use of alien species for wheat improvement, especially thosecontaining St or E in their genome components.     

Genetic Relationships Among Five Basic Genomes St, E, A, B and D in Triticeae Revealed by Genomic Southern and in situ Hybridization

The St and E are two important basic genomes in the perennial tribe Triticeae （Poaceae）. They exist in many perennial species and are very closely related to the A, B and D genomes of bread wheat （Triticum aestivum L.）. Genomic Southern hybridization and genomic in situ hybridization （GISH） were used to analyze the genomic relationships between the two genomes （St and E） and the three basic genomes （A, B and D） of T. aestivum. The semi-quantitative analysis of the Southern hybridization suggested that both St and E genomes are most closely related to the D genome, then the A genome, and relatively distant to the B genome. GISH analysis using St and E genomic DNA as probes further confirmed the conclusion. St and E are the two basic genomes of Thinopyrum ponticum （StStE^eE^bE^x） and Th. intermedium （StE^eE^b）, two perennial species successfully used in wheat improvement. Therefore, this paper provides a possible answer as to why most of the spontaneous wheat-Thinopyrum translocations and substitutions usually happen in the D genome, some in the A genome and rarely in the B genome. This would develop further use of alien species for wheat improvement, especially those containing St or E in their genome components.     

Molecular analysis of the D-genome of the Triticeae

Summary The chromosome of three tetraploid Aegilops L. species containing the D-genome were analyzed by in situ hybridization with a repeated DNA sequence clone pAS1 isolated from Aegilops squarrosa and observed to be D-genome specific. This sequence is found on all seven D-genome chromosome pairs of A. squarrosa and hexaploid wheat. Two distinct D-genome patterns were observed in the tetraploid species. The D-genome of A. cylindrica was similar to hexaploid wheat. Seven pairs of chromosomes having large amounts and numerous sites of the sequence were observed. Five chromosome pairs with fewer and smaller sites of the repetitive sequence were observed in the D-genomes of A. crassa and A. ventricosa. In addition to these major repeated sequence differences, chromosomal modifications appear to have occurred between T. aestivum and A. cylindrica and between A. crassa and A. ventricosa resulting in changes with respect to location of the sequence between the respective species. D-genome divergence with respect to pAS1 sequence appears to have occurred at least in two forms, one characterized by the changes in amount of repetitive sequence and the second by changes in location of the sequence.     

两种非放射性标记方法在染色体原位杂交中敏感性的比较

通过原位杂交比较了地高辛配基和生物素标记探针,检测染色体单拷贝基因的敏感性。结果表明：在打点检测条上地高辛配基可检出３０ｆｇ低限探针ＤＮＡ,生物素为１ｐｇ。经原位杂交地高辛配基可检测出单拷贝基因,生物素未成功。     

Improvement of Nonradioactive DNA in Situ Hybridization

With the introduction of microwave pretreatment, the quality of nonradioactive in situ hybridization (NISH) using DNA probes on formalin fixed tissue has significantly improved. Even after microwave treatment, however, there are cases where NISH results remain unsatisfactory. Therefore, we tried to improve NISH by testing other buffer systems as alternatives to the citrate buffer that is routinely applied during microwave pretreatment. By using buffer systems originally designed for immunohistochemistry, we significantly improved our NISH results. Difficult tissue samples were more accessible to NISH using these alternative buffer systems and made the quantitative evaluation easier. These results may also be of interest for combined applications of NISH and immunohistochemistry.     

体外诱导分化神经细胞的原位分子杂交方法

经１×１０－６ｍｏｌ／Ｌ视黄酸诱导的Ｐ１９细胞体外可向神经方向分化,接种于多聚赖氨酸（ｐｏｌｙＤｌｙｓｉｎｅ）和纤连蛋白（ｆｉｂｒｏｎｅｃｔｉｎ）包被的玻片后,细胞逐渐聚集成团,此时细胞的贴壁性较差,进行原位分子杂交时容易脱落。我们尝试在细胞表面覆盖一层明胶,减少了细胞的脱落,又比较了蛋白酶Ｋ和胃蛋白酶对细胞蛋白质的消化作用,确定胃蛋白酶可较温和地消化细胞蛋白质,使探针有效地透入结合,杂交后细胞亦能较完整地保留于玻片上。     

Nonradioactive in Situ Hybridization with Digoxigenin Labeled DNA Probes

Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate.     

杀配子染色体在构建小麦族缺失图谱方面的应用

山羊草属的某些染色体,当其以单价体形式附加到各种不同的基因型的普通小麦中时,就会发挥完全的或不完全的杀配子作用。在不完全的这种杀配子作用下,可以高频率的诱导产生各种类型的染色体结构畸变,如易位、缺失等,实现外源有益基因向小麦的渗入,也有利于构建缺失图谱和物理图谱［1］。本文综述了利用杀配子染色体构建小麦、黑麦、大麦染色体的缺失图谱的研究进展。     

RNA原位杂交技术的一些应用技巧

目的:检测基因在动物组织或细胞中的时空表达模式。方法:转录反义RNA探针;利用RNA原位杂交技术检测人和小鼠牙原基中若干基因的表达。结果与结论:通过优化条件,转录出完整的反义RNA探针,并成功地利用RNA原位杂交技术在组织中检测到基因的表达;分析了一些在RNA原位杂交的过程中可能碰到的问题及其解决方法。     

In Situ Hybridization of Lilium Whole Mount Synaptonemal Complex Chromosomal Preparations

Whole mount meiotic preparations of the synaptonemal complex complement of Lilium have been used for in situ hybridization experiments. A probe of the maize ribosomal DNA gene cluster has been successfully hybridized to the lily preparations. Three strong signals, corresponding to the three known lily nucleolus organizer regions, have been seen in most of the chromosome preparations. In situ hybridization experiments using meiotic preparations should be useful for identifying specific chromosomes, and for investigating the role of particular DNA molecules important to meiotic function.     

玉米两个RFLP标记的原位单杂交与共杂交定位的比较

ＲＦＬＰ标记ｂｎ１８．２３和 ｕｍｃ１１１位于玉米遗传日第 ４连锁群近端,彼此密切连锁但次序尚未确定。用生物素标记对它们进行了原位单杂交和共杂交的比较定位。在植物中,这类原位共杂交的研究为首次报道。在单一探针的原位杂交中 ｕｍｃ１１１被定位在第 １、 ４和９染色体长上,与着丝粒的百分距离分别是７．３６±２．６５、６３．６７±１．０７、４７．８７±２．９０。ｂｎ１８．２３被定位在第４和８染色体长臂上,与着丝粒的百分距离分别是８７．４２±２．４５和２７．６０±１．７５,清楚地表明了这两个标记在第４染色体上的次序。ｂｎ１８．２３和ｕｍｃ１１１分别与编码过氧化氢酶的ｃａｔ３基因和编码丝氨酸／苏氨酸蛋白激酶的ｃｄｅ２Ａ基因紧密连锁。根据供试ＲＦＬＰ标记检出位点推断了基因ｃｄｃ２Ａ和ｃａｔ３的物理位置。原位共杂交在第 ４染色体长臂上同时显示出了ｕｍｃ１１１和ｂｎ１８．２３两个标记的杂交信号,它们的位置分别与单一探针原位杂交的位置基本吻合。这为低拷贝或单一拷贝等小片段ＤＮＡ物理定位的可靠性以及它们共杂交的可行性提供了令人信服的证据。     

Identification of Alien Chromatin and Ribosomal DNA in Wheat by in Situ Hybridization

In situ hybridization was carried out to somatic cells of hexaploid Triticale “Badger”, lB/IR translocation line “Ning 8026” and IR(ID) substitution line “84056-1-36-1” using biotin-labelled total rye genomic DNA and wheat rDNA as probes, the results were as follows: 1. The probe containing the total genomic DNA from rye hybridized to the entire length of all rye chromosomes, as a result of the formation of a brown precipitate over the sites of hybridization, the rye chromosomes could be distinguished from wheat chromosomes counterstained by Wright’s solution, the distinguishable appearance of the wheat and rye chromosomes resulted in an efficient method of detecting rye chromosome or segments in wheat. 2. When the probe PTA 71 containing wheat ribosomal DNA was used to hybridize to somatic chromosomes of "Badger" and “84056-1-36-1”, six signals in “Badger” and eight in “84056-1-36-1” were observed on lB, 6B, 1R and SD, among which lB and 6B showed large in situ signals corresponding to many copies of the genes. 3. The expression behavior of wheat rDNA was found in interphase cells by in situ hybridization.     

The physical location of fourteen RFLP markers in rice (Oryza sativa L.)

A biotin-labeled in situ hybridization technique was used in order to physically map RFLP markers to the chromosomes of rice (Oryza sativa L.). Fourteen RFLP markers, associated with the ends of the linkage groups on rice chromosomes 7, 8, 11, 12, were physically mapped onto specific regions of the chromosomes. The average detection rate of in situ hybridization was 5.91%. The markers were located on seven different chromosome arms. Ten of the fourteen markers were distributed near the chromosome ends. This demonstrated that the RFLP linkage groups involved covered a wide physical distance and that the centromeric region was bisected by all but one linkage group. Two markers covered a short genetic distance but were physically distant, while two covering a longer genetic distance were physically closer together. This indicates that considerable variation can, and does, exist between genetic and physical maps.This paper is a contribution of the U.S. Department of Agriculture, Agricultural Research Service, and Missouri Agricultural Experiment Station, Journal Series No. 11 882All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicapThis paper reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the U.S. Department of Agriculture or the University of Missouri     

荧光原位杂交技术及其在植物研究中的应用

荧光原位杂交(FISH)是20世纪生物学领域的一项新技术。FISH应用细胞遗传学和分子生物学的基本原理,作为架设细胞遗传学与分子生物学之间的桥梁,现已被广泛应用于植物学各方面的研究。本文就FISH的基本原理、技术发展及其在植物遗传育种、起源进化、染色体物理图谱构建方面的应用及发展趋势进行了综述。     

Temporal Expression of Mouse Glial Fibrillary Acidic Protein mRNA Studied by a Rapid In Situ Hybridization Procedure

A rapid and sensitive in situ hybridization technique is described for the detection of mRNA sequences in 6-8-micron cryostat sections. The method incorporates the use of alpha-thio-35S-labelled nucleoside triphosphates for the generation of high-specific-activity DNA probes and a high-stringency washing procedure that virtually eliminates background without unduly compromising histological integrity. Whereas signal resolution is less than that observed using 3H probes, 35S-labelled probes are well-suited for experiments where resolution at the cellular level is required. The method has been applied to a study of the developmental regulation of glial fibrillary acidic protein (GFAP) mRNA expression in developing mouse brain. GFAP-specific sequences are first detectable after the second postnatal day, and thereafter rise to a level that is maintained throughout development and into adulthood. The distribution of GFAP-encoding sequences broadly reflects the known distribution of astrocytes, but the levels of mRNA within these cells vary by a surprisingly large amount depending on their location. For example, in adult animals, the astrocytes of the glial limitans contain an abundance of GFAP-specific mRNA that is higher than corresponding levels in astrocytes in the cerebellar white matter, whereas these cells in turn contain considerably more GFAP-specific mRNA than astrocytes in the gray matter of the cerebrum. Unexpectedly, parallel RNA blot transfer experiments show the existence of some GFAP-encoding mRNA size heterogeneity that is restricted to the first postnatal week.     

RNA原位杂交实用技术

利用互补RNA为探针进行原位杂交是分析组织或细胞内RNA分布的行之有效的方法 ,通过对mRNA分布的研究可以了解特定基因的表达情况。原位杂交技术过程较长 ,操作繁琐 ,从而在一些实验中不能得到很好的使用 ,为此本文根据我们过去的实际操作经验对该技术中的一些使用技巧作简要的介绍。